Diversification of the Translational Science workforce is a strategic goal for the National Center for the Advancement of Translational Science (NCATS) program. NCATS has identified the development of translational science education, training, and support for a diverse translational science workforce as key to advancing the growing field of translational science. An annual mixed-methods assessment has been conducted on Common Metrics data submitted by over 60 Clinical & Translational Science Awards (CTSA) programs nationwide and includes metrics addressing recruitment and retention of scientists with particular attention to underrepresented persons and women. This article describes a methodology for the development of From Insights to Action, a resource for guiding program implementation and strategic planning to develop a diverse clinical and translational science workforce. This was informed by the Common Metrics Initiative process and constituted of findings from qualitative interviews of a subset of CTSAs that participated. The dissemination of this guide had several impacts, including providing structural foci for the CTSA Fall 2020 program meeting centered on Diversity, Equity, and Inclusion in translational science; addressing NCATS’ goal of workforce diversity; and understanding the number of diverse graduates still engaged in research.

OBJECTIVES/GOALS: The first aim was to construct a controlled and high resolution FUS water tank characterization system with 1 micron step-sizes. The second aim was to create two unique standardized protocols for mapping the generated acoustic field from FUS transducers; protocol one maps the full 3D field while protocol two rapidly detects changes to the original plot. METHODS/STUDY POPULATION: To accomplish aim one, the focused ultrasound mapping platform was constructed with a water conditioning unit for water degassing and temperature control, a three-axis stage with 1 micron step-size capabilities, and a data plotting software. To measure the outcomes of aim one, the water temperature was monitored, and axis step sizes were measured through ten independent axis translation recordings. To accomplish aim two, FUS acquisitions were executed at different resolutions. For FUS localization at the cellular level, a 1-5 micron step size is required. Once the initial scan was performed, duplicate scans were executed to detect inherent perturbations or errors in the system. Once calculated, the best methods of detecting true changes to FUS signals are proposed. RESULTS/ANTICIPATED RESULTS: The FUS characterization system maintained water temperature and performed 1 micron step-sizes. While pre-existing platforms have demonstrated a resolution of one thousand recordings per cubic millimeter, the proposed system (time and computing power willing) can record one billion recordings per cubic millimeter. In practice, a resolution of 20 micron was sufficient for non-cellular level FUS characterizations. Successive 2D scans were reliably stacked to form a 3D rendering of the generated acoustic field with the average focal point intensity yielding a 1% coefficient of variation between identical scans. This inherent variation can be used as the threshold of significance for true change detection; to rapidly detect changes to the FUS signal, sampling can be performed at regions of high baseline values. DISCUSSION/SIGNIFICANCE: Focused ultrasound medical devices are gaining popularity for treatments including tumor ablation, neuromodulation, and drug delivery; however, the field lacks a standardized method to characterize these FUS transducers. The presented platform and protocols enable a rigorous and high quality translation through verification and validation.

Objectives/Goals: To determine the acceptability of a mobile/school-based diabetes and prevention clinic to overcome transportation barriers and improve attendance at endocrinology appointments for youth with type 1 diabetes, type 2 diabetes, MODY diabetes, and obesity living in socially vulnerable communities Methods/Study Population: This study utilized a 3-phase implementation process focused on feasibility, demand, and acceptability. Phase 1: identified high-need areas using patient volume and disease control metrics. Collaborated with school districts to define needs, enrolled them through a memorandum of understanding, and subsequently recruited and consented existing patients attending these schools. Phase 2 piloted the program to refine logistics and workflows. Semi-structured interviews and surveys were conducted with caregivers to assess program acceptability and satisfaction. Phase 3 is ongoing and will track clinical outcomes including glycemic control, appointment attendance, hospital admissions, emergency department visits, and self-management metrics. Results/Anticipated Results: Demand for the program was high with 91% (10/11) of approached districts agreeing to participate. District enrollment was staggered, starting with 1 district in August 2023, expanding to 5 in September, 7 in October, and reaching 8 districts by May 2024. From August 2023 to July 2024, the school-based mobile clinic conducted 355 individual clinical encounters addressing diabetes and obesity. The encounters included 180 for type 1 diabetes, 69 for type 2 diabetes, 8 for MODY diabetes, and 98 for obesity. Interviews and surveys with 36 caregivers are ongoing to evaluate program satisfaction. Discussion/Significance of Impact: The data suggest that this innovative healthcare delivery model is feasible. District-level enrollment demonstrates a demand for the program. Results from the interviews and surveys will further characterize the program’s acceptability among caregivers and lay the groundwork for future efficacy testing.

OBJECTIVES/GOALS: The innate immune responses to Multisystem Inflammatory Syndrome in Children (MIS-C) are not fully known. Using samples from MIS-C, we will assess the cellular responses and develop a novel Tri-Specific Killer Engager (TRiKE) that engages innate immune cells to improve those responses. METHODS/STUDY POPULATION: We collected blood samples from 60 pediatric patients from which we isolated plasma and peripheral blood mononuclear cells. We received blood samples from 13 MIS-C, 32 severe acute COVID, 5 COVID-19 asymptomatic, and 15 COVID-19 negative patients. Using plasma, we then performed ELISAs to determine IgG antibody levels against SARS-CoV-2 and plaque reduction neutralization tests to determine neutralizing antibody functions. We isolated DNA to look at Fc receptor genetics. We also utilized utilize flow cytometry assays determine the phagocytosis and killing abilities of the innate cells from these patients. This data will be correlated with clinical outcomes. Additionally, we have developed a novel SARS-CoV-2 TRiKE which directs natural killer (NK) cell killing specifically to of COVID-19 infected cells. RESULTS/ANTICIPATED RESULTS: MIS-C patients had higher IgG antibody titers against SARS-CoV-2 compared to children with symptomatic or asymptomatic COVID. MIS-C patients also neutralized SARS-CoV-2 more effectively than children with acute symptomatic or asymptomatic COVID-19. We found natural killer cells and monocytes are dysfunctional in MIS-C patients and do not kill SARS-CoV-2 infected cells as well. Specifically, NK cells do not kill COVID-19 infected cells as well. To combat this, we have successfully generated and are now testing a Tri-Specific Killer engager (TRiKE) which binds one ends to NK cells, one end to the Spike protein on COVID-19 infected cells and contains IL-15 to improve NK cell function. We anticipate that we can improve NK cell killing of COVID-19 infected cells with this TRiKE. DISCUSSION/SIGNIFICANCE: We found that MIS-C patients have antibodies that can neutralize SARS-CoV-2 but that that innate immune cells that engage antibodies are dysfunctional. We are have successfully developed and are targeting this response with a TRiKE to improve innate immune cell functional; this may serve as an adjunctive therapeutic if proven successful.

OBJECTIVES/GOALS: Precision care may engage smokers and providers in treatment but is understudied in the community. We piloted guideline-based care (GBC) alone or with Respiragene, a lung cancer polygenic risk score (PRS, 1-10), or metabolism-informed choice of medication using the nicotine metabolite ratio (NMR). METHODS/STUDY POPULATION: Daily smokers (n = 58) with stored biospecimens in the Southern Community Cohort Study were randomized 1:1:1 to GBC, PRS, or NMR, counseled to quit smoking, and co-selected FDA-approved cessation medication (nicotine replacement, varenicline) with a tobacco counselor. In PRS, precision motivational counseling was guided by PRS (i.e., lung cancer risk 10-40-fold that of never-smokers). In NMR, precision medication recommendations consisted of varenicline for faster metabolizers (NMR≥0.31) and nicotine replacement for slow metabolizers (NMR<0.31). Feasibility was defined as achieving at least 50% provider engagement (med prescription) and at least 50% patient engagement (self-reported med use). RESULTS/ANTICIPATED RESULTS: Participants were median age 59, 72% female, 81% Black, 60% with incomes <$15,000; median cigarettes/day was 15 (IQR 8-20) and 52% reported time-to-first cigarette <5 minutes, illustrating moderate nicotine dependence. Providers confirmed medication prescriptions for 40% of patients (32% GBC, 50% PRS, 37% NMR) and 83% of patients reported using medication (prescribed or unprescribed) during the study (90% GBC, 80% PRS, 79% NMR). At 6-month follow-up, 27% (n = 15) reported cessation (39% GBC, 16% PRS, 26% NMR). Among persistent smokers, 46% reported smoking at least 50% fewer cigarettes/day compared to baseline (45% GBC, 38% PRS, 57% NMR). Small sample size precluded statistical comparisons. DISCUSSION/SIGNIFICANCE OF IMPACT: Precision interventions to quit smoking are feasible for community smokers, who engaged at high rates. However, only 40% of providers supported patients’ quit attempts with medication prescriptions. Future research should test strategies to raise provider engagement in precision smoking treatment. CONFLICT OF INTEREST DESCRIPTION: R.F.T. has consulted for Quinn Emmanual and Apotex on unrelated topics. H.A.T. reported providing input on design for a phase 3 trial of cytisine proposed by Achieve Life Sciences and being a principal investigator of National Institutes of Health–sponsored studies for smoking cessation that include medications donated by the manufacturers. Other authors declare no potential conflicts of interest.

OBJECTIVES/GOALS: One in six children in the U.S. has a Neurodevelopmental Disability (NDD). Prechtl’s General Movement Assessment (GMA) is a qualitative predictor of early motor dysfunction. However, no quantitative biomechanical assessment exists to more accurately identify all patients with NDD. METHODS/STUDY POPULATION: With UAMS IRB approval, as part of a larger study, healthy infants were filmed while lying supine on a force plate for 2 minutes. We studied 12 healthy full-term infants (gestational age: 38.9±1.5 weeks, age: 2.1-7.0 months; 7M, 5F; length: 64.0±5.2 cm; weight: 7.2±1.3 kg). Within our data set there were 3 infants transitioning to fidgety period (≤3 months), 4 in the fidgety period, (3-5 months), and 5 that matured beyond fidgety period (>5 months). Center of pressure (COP) path-lengths were gathered from the force plate at 1000 Hz. We grouped our data with K-means clustering and performed statistical analysis with ANOVA. RESULTS/ANTICIPATED RESULTS: We divided our data into 3 distinct clusters. The first group contained infants with moderate variability of movements which included 2 infants between 3 and 5 months and 2 infants slightly outside of this range. The second group, with mild variability in movements, included 4 infants between 2 and 3 months as well as 2 infants just older than 5 months. The third group, with little variability in movements, included 2 infants older than 5 months. A GMA reader (TJ) qualitatively confirmed these findings with video footage. Using a threshold of p<0.05, data sets within the clusters were similar and significantly different from other clusters. DISCUSSION/SIGNIFICANCE OF IMPACT: Fidgety infants have greater variability in COP patterns than their mature counterparts. We anticipate additional COP measurements will correspond with qualitative GMA analyses. Artificial Intelligence-based quantification of the GMA may be useful in earlier detection or prediction of NDD outcomes.

OBJECTIVES/SPECIFIC AIMS: This study aims to understand the utility of 5-ALA and SWIG in detecting areas of neoplasm in a murine model of GBM. Primary outcome is the distribution of the two dyes in comparison to the true tumor extent; the sensitivity, specificity, PPV, and NPV of both dyes will be calculated. The secondary outcome is the suitability of existing cell-lines used for GBM research for studies in fluorescence-guided surgery. METHODS/STUDY POPULATION: Two cell lines are used for this research: U87, derived from human GBM; and GL261, derived from rodent stem cells. U87 are implanted intracranially into 6-week old athymic, nude, female mice, while GL261 are implanted intracranially into 10-week old female C57BL/6 mice. The mice are weighed every 3 days to monitor health and bioluminescence imaging is performed between 7-10 days after implantation to confirm tumor implantation and monitor tumor growth. The mice are sacrificed between 10-21 days after implantation. 5mg/kg of intravenous ICG is administered 24-hours prior to harvest and 250mg/kg of intraperitoneal 5-ALA is administered 3-hours prior to harvest. Once the mice are sacrificed, their brains are quickly harvested and placed in cold formalin. Using a high-resolution Odyssey CLx scanner, near-infrared fluorescence from ICG is captured in coronal cross sections of the brains through the tumor. Similarly, 5-ALA fluorescence is imaged using a 405nm LED excitation source and 610-690nm bandpass filter. Afterwards, slices of the brain are stained with H&E, which serves as the gold-standard of the extent of tumor. Images from ICG, 5-ALA, and H&E can then be compared using ImageJ to compare the extent of tumor to the distribution of the dyes. RESULTS/ANTICIPATED RESULTS: In separate, previous studies in humans, both 5-ALA and SWIG have demonstrated utility in detecting residual neoplasm in HGG resections. In general, 5-ALA is more specific for areas of neoplasm, while SWIG is more sensitive. Thus, I anticipate that in this study, SWIG will show a greater distribution than 5-ALA, with SWIG distributing to areas beyond the tumor and 5-ALA distributing within, but not completely covering, the tumor. SWIG’s sensitivity and NPV for detecting tumor should be >90%, while its specificity and PPV may be closer to 50%. For 5-ALA, specificity and PPV should be close to 80-90%, but its sensitivity and NPV may be <50%. In terms of cell-line, preliminary results suggest that U87 cells are not suitable for research involving 5-ALA. We suspect that this is partly due to the limited infiltrative nature of U87 cells; in fact, the cells form a spherical mass, imitating metastases rather than true HGGs. The U87 masses do not have significant vascularity, which likely limits the amount of 5-ALA that can distribute to inside the tumor. DISCUSSION/SIGNIFICANCE OF IMPACT: 5-ALA is currently the only FDA-approved agent for fluorescence-guided neurosurgery. However, it has multiple limitations, which ultimately results in its low sensitivity and NPV. Our novel technique, which has demonstrated much higher sensitivity at the cost of specificity, offers an alternative that may help surgeons better achieve total resections in the operating room. These two agents have not been compared directly in humans or mice. Thus, this experiment sets up an important precedent, on which a human clinical trial comparing the two agents’ effects on resection rates and patient outcome can be performed. Ultimately, this work will lay the foundation for future research into fluorescence-guided neurosurgery, both in the visible and NIR spectrum.

Objectives/Goals: To develop an information-theoretic alternative to ALE that integrates uneven datasets using log-likelihood ratios (LLRs) under noise versus signal world models. This approach aims to improve reproducibility, signal-to-noise ratio, and computational scalability in neuroimaging meta-analysis. Methods/Study Population: We used both real and simulated coordinate-based datasets to build an information-theoretic meta-analysis framework. The method models each study’s activations under noise versus signal expectations and computes voxelwise LLRs to combine information asymmetrically across uneven datasets. Implemented in Python with NumPy and custom modules, it integrates watershed segmentation for cluster detection and simulation-based calibration. Validation compares results to ALE, testing robustness to injected noise and recovery from highly imbalanced datasets. Results/Anticipated Results: Studies are ongoing. We anticipate improved SNR and recovery robustness compared to ALE, as our approach avoids pre-thresholding before combining information across datasets. Simulation evidence suggests far greater computational efficiency (e.g., reducing 45-min–20-hour ALE runs to under two minutes locally). Conceptually, the framework parallels modern AI training strategies – modeling signal and noise distributions to calibrate evidence rather than relying on fixed significance thresholds. These innovations leverage information theory, watershed algorithms, and simulation calibration to yield interpretable, high-resolution evidence maps. Discussion/Significance of Impact: This framework advances coordinate-based meta-analysis by modeling evidence directly rather than inferring it through p-values. It improves reproducibility, interpretability, and cross-disorder comparability, supporting more robust and scalable neurocircuit mapping for researchers and clinicians.

ABSTRACT IMPACT: The pathophysiologic features of a metabolomic endotype that predicts patient outcomes due to sepsis have the potential to direct new therapies that target immune dysregulation and bioenergetic insufficiency. OBJECTIVES/GOALS: Acute respiratory failure (ARF) requiring mechanical ventilation is a frequent complication of sepsis and other disorders. It is associated with high morbidity and mortality. Despite its severity and prevalence, little is known about metabolic and bioenergetic changes that accompanying ARF. METHODS/STUDY POPULATION: In this study, semiquantitative and quantitative ultrahigh performance liquid chromatography mass spectrometry (UHPLC MS) analysis was performed on patient serum collected from the Trial with Acute Respiratory failure patients: evaluation of Global Exercise Therapies (TARGET). Serum from survivors (n=15) and nonsurvivors (n=15) was collected at day 1 and day 3 after admission to the medical intensive care unit as well as at discharge in survivors. Pathway analysis of the biochemical changes was performed to determine whether the disruption in specific metabolic pathways can identify the bioenergetic and metabolomic profile of these patients. RESULTS/ANTICIPATED RESULTS: Significant metabolomic differences were related to biosynthetic intermediates of redox cofactors nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP), increased acyl-carnitines, and decreased acyl-glycerophosphocholines in nonsurvivors compared to survivors. The metabolites associated with poor outcomes are substrates of enzymatic processes dependent on NAD(P), while the abundance of NAD cofactors rely on the bioavailability of dietary vitamins B1, B2 and B6. Changes in the efficiency of the nicotinamide-derived cofactors’ biosynthetic pathways also associate with an alteration of the glutathione-dependent drug metabolism as characterized by the substantial differences observed in the acetaminophen metabolome. DISCUSSION/SIGNIFICANCE OF FINDINGS: This metabolomic endotype represents a previously unappreciated association between severity of outcomes and micronutrient deficiency, thus pointing to new pharmacologic targets and highlighting the need for nutritional remediation upon hospitalization to improve patient outcomes due to ARF.

Objectives/Goals: There has been a significant increase in the engagement of researchers with persons with lived and living experience, driven by a growing recognition of the invaluable insights and expertise these individuals bring to the research process. However, there remains a need for research teams to learn how to engage with patients and community partners. Methods/Study Population: The EMPOWER project is a collaborative of members with lived/living expertise of substance use during pregnancy. EMPOWER meets three times per month in support of their goals of patient-centered research, changing the narrative through conversations, and building the capacity of persons with lived experience to co-lead research. Through over 4 years of active collaboration and co-learning, EMPOWER has identified and created methods that support meaningful patient engagement and capacity building. Results/Anticipated Results: EMPOWER has identified the following methods that support meaningful engagement and capacity building: Mind-Mapping Exercises, Personal and Collective Journey Mapping, 8-month research training curriculum, Digital Storytelling, Learning Labs, and World Cafe Meetings. These methods have led to a collaborative of 20 individuals with lived experience of substance use to identify research gaps, prioritize research questions, design data collection tools, analyze research data, disseminate research results, and consult with healthcare teams in efforts to improve clinical care. To-date EMPOWER have given over 10 invited presentations and is currently writing manuscripts. Discussion/Significance of Impact: Meaningful patient and community engagement is critical to developing and implementing healthcare interventions. Unfortunately, engagement efforts are often lack in building capacity of patients and community, which impedes their ability to fully engage in all phases of research.

OBJECTIVES/GOALS: The goal of this project was to perform an exploratory analysis of the research themes of scientific publications from the Miami Clinical and Translational Science Institute using text mining techniques and using bibliometric characterization and network analysis to further assess research trends. METHODS/STUDY POPULATION: Publications were identified from the Web of Science database using Miami CTSI grant numbers as search criteria for the period 2013-2021 and KL2 scholar publications. Following data pre-processing, topic modeling was performed using the Latent Dirichlet Allocation algorithm and cluster analysis in the R programming language. The resulting themes will be further analyzed by employing a citation-based impact assessment approach to identify trends over time. Network analysis of publications will be performed using the VOSviewer package to visualize publication networks using citation and co-authorship relations within each major theme and their evolution over time. Findings will be evaluated for alignment with institutional research strategy. RESULTS/ANTICIPATED RESULTS: About 400 CTSI publications from 2013-2021 to date were used for analysis. Twenty topics and five major research themes were identified among the Miami CTSI publications – neuroscience, cancer, community and public health, metabolics, and HIV/infectious diseases. Top keywords within each topic were aligned with the most frequent author-assigned keywords for that topic. The CTSI research themes were also well-aligned with the institutional vision for research and focus areas. Trends using citations and networks for each research theme are currently being analyzed and results will be included in the overall findings post analysis. DISCUSSION/SIGNIFICANCE: Text mining was successfully used in identifying topics and research themes for clinical and translational research publications of the Miami CTSI, and in combination with bibliometric characterization, will be helpful in shaping CTSI strategy and alignment with the universitys research priorities.

OBJECTIVES/GOALS: Age-related lobular involution (LI) is a physiological process of breast epithelial regression that occurs primarily during perimenopause (ages 45-55); women in this age range for which the process of LI is delayed, defined as LI stagnation, show significantly increased risk of breast cancer as compared to LI progression patients. METHODS/STUDY POPULATION: The Mayo Clinic Benign Breast Disease (BBD) cohort includes ~1000 women who had multiple sequential benign biopsies. 103 patients were found to have sequential biopsies during the perimenopausal period, of which 10 eventually progressed to breast cancer. These patients were assessed for LI stagnation vs LI progression by quantifying 10 lobules per slide and comparing median acini number and median lobule size between initial and subsequent biopsies from the same patients. RNA was derived from whole tissue sections from the initial biopsies, and profiled using NanoString IO360 and BC360, which were normalized using RUVg methods. Differentially expressed genes associated with LI stagnation were defined as having two-tailed, unpaired p-values less than 0.05. RESULTS/ANTICIPATED RESULTS: Analysis showed subsetting patient sets by time between biopsies improves classification of stagnant vs. progression. Differential gene analysis identified 37 genes associated with LI stagnation and LI progression, and 20 of these genes were found to overlap a set of 128 gnese that were differentially expressed between women who subsequently developed breast cancer vs remained cancer-free. These genes represent potential biomarkers of processes that link LI stagnation and increased breast cancer risk. DISCUSSION/SIGNIFICANCE: In future studies, we intend to study these genes that were shown to be upregulated in LI stagnation for their association with subsequent development of breast cancer in independent cohorts of women with BBD. We will use this knowledge to improve individualized risk assessment, which will help focus surveillance and prevention strategies.

OBJECTIVES/GOALS: Weaimed to compare subjective and objective sleep in individuals with chronic post-concussive symptoms. We hypothesized an association between self-reported sleep quality and objective sleep parameters, which is different for concussed and control cohorts. METHODS/STUDY POPULATION: 28 individuals with chronic post-concussive symptoms and 13 age-matched controls (no concussion history) completed the ISI, PSQI, PROMIS Depression, Anxiety, Stress and Cognitive questionnaires at enrollment. Objective sleep parameters were obtained for a minimum of 7 days and up to 30 days with a validated sleep monitoring device placed under the subject’s bed (Emfit). For each night, raw activity data per minute were analyzed to determinein-bed, sleep, wake, andout-of-bedtimes. These measures were used to calculate total sleep time (TST), sleep onset latency (SOL), and wake after sleep onset (WASO) for each night. RESULTS/ANTICIPATED RESULTS: Concussed individuals reported worse sleep with PSQI and ISI scores significantly higher than controls. They also showedsignificant associations between PSQI and Dep ression, ISI and Depression, and ISI and Anxiety scores. There was no difference between objective sleep parameters in the concussed and control cohorts (in-bed/sleep/wake/out-of-bed times, TST, SOL, and WASO). Instead, higher PSQI, ISI, Depression, Anxiety, and Stress scores (greater symptom burden) were all associated with later sleep times, where as higher Cognitive scores (greater cognitive function) were associated with earlier sleep times, regardless of group status. DISCUSSION/SIGNIFICANCE: Concussed individuals report worse subjective sleep but no differences to controls when objectively assessing sleep. Depression/anxiety, and not concussion status, determine objective sleep parameters. Psychiatric comorbidities should inform the treatment of post-concussive sleep disturbances.

OBJECTIVES/GOALS: We designed the Biocascade Exhaled Breath Sampler (BEBS) to characterize viral aerosol shedding among individuals with influenza and other respiratory virus infections. We first aimed to test the BEBS on volunteer COVID-19 cases and report the aerodynamic size distribution of exhaled breath aerosol particles carrying SARS-CoV-2 RNA. METHODS/STUDY POPULATION: From June 15 through December 15, 2022, we recruited 27 PCR-confirmed COVID-19 cases from a college campus and the surrounding community to provide 30-minute breath samples into a well-validated Gesundheit-II (G-II) exhaled breath aerosol sampler. Among these individuals, 17 provided an additional exhaled breath sample into the newly designed BEBS. We quantified samples for viral RNA using reverse transcription digital polymerase chain reaction (RT-dPCR) and determined the viral RNA copies collected within two aerosol size fractions (≤5 µm and >5 µm in diameter) from the G-II, and four aerosol size fractions (<1.15 µm, 1.15–3.2 µm, 3.3–8.2 µm, and >8.2 µm) from the BEBS. RESULTS/ANTICIPATED RESULTS: Individuals with a SARS-CoV-2 Omicron BA.4 or BA.5 infection shed virus in aerosols at an average rate of 7.5x103 RNA copies per 30-minute G-II sample, with 78% of the total RNA in aerosols ≤5 µm in diameter. Among the BEBS samples, 10% of the total viral RNA was detected in aerosols <1.15 µm, 43% in 1.15–3.2 µm, 37% in 3.3–8.2 µm, and 10% in the >8.2 µm size fraction. Based on viral RNA loads, our results indicate that exhaled aerosols ≤3.2 µm contribute the majority of SARS-CoV-2 inhalation exposure. DISCUSSION/SIGNIFICANCE: Our data provide additional evidence that respirable aerosols contribute to the spread of SARS-CoV-2. Thus, our data suggest that mitigation measures designed to reduce infectious aerosol inhalation, such as ventilation and the use of air cleaners and respirators, are needed to control the spread.

Objectives/Goals: To evaluate whether signup recency in the Indiana CTSI All IN For Health Volunteer Registry influences responsiveness to outreach for clinical trial recruitment, testing the hypothesis that newer records are more likely to yield referrals. Methods/Study Population: Outreach data from the TrialX Connect registry were analyzed. Each outreach was coded as referral versus no response. Volunteers were grouped by time since signup: <6 months (n=386), 6–12 months (n=282), 1–2 years (n=659), 2–3 years (n=760), and >3 years (n=13,592). Conversion rate was defined as referrals per unique volunteers outreached. Statistical comparisons were made across categories. Results/Anticipated Results: Conversion rates were stable across categories (2.14–3.36%). The 6–12 month cohort had the highest rate (3.36%), while 2–3 years had the lowest (2.14%). Newer (<6 months) volunteers converted at 2.28%. Most referrals (n=345) came from >3 year signups, reflecting their numerical dominance (13,592 signups, ~90% of registry). Discussion/Significance of Impact: Signup recency showed little impact on responsiveness (steady at ~2–3%). Older records produced most referrals due to larger volume, emphasizing the lasting value of long-term participants. Results suggest focusing on sustained engagement and retention over simply expanding the registry.

OBJECTIVES/SPECIFIC AIMS: The primary objective of this study was to evaluate the performance of a bone fracture targeted systemically administrable bone anabolic as a potential therapeutic for bone fracture repair. Currently all bone fracture repair therapeutic require local administration during surgery. However, the population that need the most assistance in repair bone fractures are not eligible for surgery. So, it was our goal to design an inject-able therapeutic to assist in bone fracture repair to reduce the invasiveness. The injectable nature of it allows for repair administration of the bone anabolic and for therapeutic effect throughout the entire bone fracture healing process. Targeting it to the bone fracture site reduces the toxicity and increases the efficacy. METHODS/STUDY POPULATION: METHODS To achieve the above objective, a bone mineral-(hydroxyapatite-) targeting oligopeptide was conjugated to the non-signaling end of an engineered parathyroid hormone related protein fragment 1-46 with substitutions at Glu22,25, Leu23,28,31, Aib29, Lys26,30 (ePTHrP). The negatively charged oligopeptide has been shown to target raw hydroxyapatite with remarkable specificity, while the attached PTHrP has been demonstrated to induce sustained and accelerated bone growth under control of endogenous morphogenic regulatory factors. The conjugate’s specificity arises from the fact that raw hydroxyapatite is only exposed whenever a bone is fractured, surgically cut, grafted, or induced to undergo accelerated remodeling. The hydroxyapatite-targeted conjugate can therefore be administered systemically (i.e. without invasive surgery or localized injection) and still accumulate on the exposed hydroxyapatite at the fracture site where it accelerates the healing process Murine in vivo experiments were conducted on female Swiss Webster mice (10 per group). Femoral fractures were induced with a 3-point bending device and stabilized. Mice were dosed with 3 nmol/kg/d of targeted-ePTHrP, non-conjugated (free) ePTHrP, or saline. Following a 4-week study, fracture callus densities were measured using microCT. Canine in vivo experiments were conducted on 1-year-old male beagles. Beagles underwent a 10 mm bilateral ulnar ostectomy. Two dogs in the treatment group and Three dogs in the control group were dosed daily with either targeted-ePTHrP 0.5nmol/kg/d or saline respectively. Dogs were x-rayed weekly for the first 6 weeks and then every other week thereafter. One tailed ANOVA followed by Dunnett’s post-hoc test was used to establish significance. All animal experiments were conducted as described in approved IACUC protocols. P<0.05 was considered significant. RESULTS/ANTICIPATED RESULTS: RESULTS SECTION: In the murine studies we observed a marked increase in fracture callus size and a 2-fold increase in bone deposition was observed in the targeted-ePTHrP group over the saline group (P<0.01). A significant doubling in bone density was also observed. Targeted-ePTHrP group fractured femurs were able to achieve their pre-fracture strength as early as 3 weeks compared to 9 weeks in the saline mice representing a 66% reduction in healing time. In the canine studies, we observe a significantly higher closure of the ostectomy gap than saline controls (P<0.05). In addition, no significant differences in weight are observed in the treatment vs. saline controls. No significant difference between the control group and treatment groups was found in a histological investigation of the organs. DISCUSSION/SIGNIFICANCE OF IMPACT: DISCUSSION: Although attempts have been made in developing a systemically administered fracture therapeutic for fracture repair, i.e. teriparatide, to date, no such anabolics have been approved for this use. In these studies there is evidence that anabolic activity was occurring at the fracture site, but at a level that did not meet FDA required end-points.2 It is plausible that if sufficient drug were to be delivered to a fracture site then improved fracture repair would be possible. In previous studies, we demonstrated fracture specific accumulation bone anabolics can be achieved by modifying the drug with acidic oligopeptides.3 Here, by modifying a safe, clinically proven, parathyroid hormone receptor agonist with an acidic oligopeptide we observe improved bone deposition and strength in mice. Furthermore, when administered to canine critical sized defect ostectomies, a more relevant and difficult model, we observe improved ostectomy closure. CLINICAL RELEVANCE:: The ability to accelerate bone fracture repair is a fundamental need that has not been addressed by conventional methods. By targeting bone anabolic agents to bone fractures, we can deliver sufficient concentrations of anabolic agent to the fracture site to accelerate healing, thus avoiding surgery and any ectopic bone growth associated with locally-applied bone anabolic agents.

OBJECTIVES/GOALS: Effective translation of data to inform real-time patient care is lacking in addiction inpatient settings. The current study presents the optimization of a Comprehensive Rehabilitation Assessment summary report that is used by clinicians to individualize treatment. METHODS/STUDY POPULATION: A multi-aim approach was taken that utilized aspects of various implementation science frameworks. Participants were clinical staff (N = 7; female = 71%). A quantitative survey was used for aims 1 and 2 to assess motives and context around the report as well as evaluate the design of it. Aim 3 focused on optimization via semi-structured interviews. Descriptive and modified content analyses were utilized appropriately for each aim. RESULTS/ANTICIPATED RESULTS: Five versions of the assessment report were created between February 2021 and August 2022, the most recent of was adapted into patients’electronic medical records based on study results. Each report version, participants’results/feedback, and researchers’perceived barriers to this translational process will be discussed. DISCUSSION/SIGNIFICANCE: The current study highlights a replicable approach for optimizing the translation of assessment data into treatment for patients with disorders of addiction.

Objectives/Goals: To date, no study has jointly profiled RNA isoforms and modifications associated with Alzheimer’s disease neuropathological changes (ADNC) in a human cohort. Therefore, the overall objective of this study is to identify and characterize RNA isoforms and modifications that associate with ADNC by leveraging long-read direct RNA sequencing. Methods/Study Population: This translational study will analyze human prefrontal cortex tissue from a well-defined cohort of 12 individuals. The cohort consists of six individuals with severe Alzheimer’s disease neuropathological changes and six age- and sex-matched non-demented controls. Leveraging long-read direct RNA sequencing, which uniquely quantifies both RNA isoform and modifications simultaneously, can provide novel insights into how severe Alzheimer’s disease neuropathological changes drive changes in RNA isoform expression patterns and alter RNA modifications contributing to isoform dysregulation. Results/Anticipated Results: My preliminary data from human prefrontal cortex (n=2) revealed pseudouridine sites, 1 of ~170 known RNA modifications, in Alzheimer’s disease-risk genes (APP, CLU, SORT1, and BIN1), supporting the need to map RNA modifications across Alzheimer’s disease-relevant isoforms. My central hypothesis is that severe Alzheimer’s disease neuropathological changes (ADNCs) drive transcriptomic and epitranscriptomic dysregulation by altering RNA isoforms. Thus, my two working hypotheses are 1) severe ADNC drives transcriptomic dysregulation of RNA isoforms and 2) severe ADNC drives epitranscriptomic dysregulation of RNA modifications. Discussion/Significance of Impact: This project will be the first to jointly profile RNA isoforms and modifications accounting for Alzheimer’s disease neuropathological changes, offering candidate regulatory targets for therapeutic development. Importantly, this research will equip me with an expertise in RNA biology, neurodegenerative diseases, and bioinformatics.